Bio-sensing requires surface functionalization to detect a particular target molecule or ion. The surface has to be uniform, consistent and should have an ability to get modified without altering the original properties with respect to a target ion or molecule to be detected. Uniformity is of utmost importance for unconstrained and clutter-free binding of the target bio-molecule; small size, non toxicity to use as a fluorescent probe. These properties are important in achieving sensitivities of the order of 10−8-10−9 M, especially in the case of piezo-electric sensors and also as a florescent chip as many metal ions plays vital role at extremely low concentrations during patho-physiology. The principal aim was dynamic utilization of noble metal particles with single domain synthesis and sample preparation.
Gold Nano Particles (AuNPs) are potential candidates for the development of nano-bio-sensors, diagnostic and therapy as they have been widely used in understanding the biological processes and also in diversified biomedical applications. AuNP's have attractive features such as, inert, easy and versatile surface chemistry to modify, biocompatibility negligible toxicity and researchers have gained enough experiences in synthesizing different size and shapes of AuNP's in controllable manner. Further, AuNP's are having very high sensitivity towards biomolecule, tuned SPR/luminescent, retention of bioactivity and opportunity for 3D imaging are important characters as a fluorescent probe/marker. Reliable and high-yielding methods for the synthesis of AuNP's, including those with spherical and non spherical shapes, have been developed over the last century. It is necessary to synthesize the nanomaterial for bioconjucation which can be universally used as a tracer as well in therapy. The very small nanoparticles, clusters and nanoroads shows visible to IR fluorescence. However small nanoparticles, clusters and nanorod's shows some form of toxicity. As the size increases due to the autocatalysis they convert to form the stable super critical nucleus showing surface plasmon; 5-25 nm particles shows the surface plasmon at 510-540 nm (Concha Tojo et al; Materials; 2011, 4, 55-72) and diminishing the fluorescent property, as the fluorescence is the function of molecule like electronic structures and quantum confinement effect which is observed in very small nanoparticles. (Tatsuya Tsukuda, Bull. Chem. Soc. Jpn., 2012, 85 (2), 151-168).
Ref made to Beatriz Santiago Gonzalez et al: ACS Nano lett. 2010, 10, 4217-4221 where Nonoclusters of gold particles capped with PVP is reported. Due to the weak bonding of PVP mass spectra only shows the peak related to Au2 and Au3 and the theoretical calculation using the Jellium model shows fluorescence emission at 293 nm and 336 nm which are related to clusters of Au2 and Au3. It showed UV (300 to 400 nm) emission from Au2 to Au11 PVP clusters however such small clusters are difficult to characterize and handle to utilize in any kind of applications. In the present invention the surface capped nano aggregates of ultra small clusters which are also stabilized by amine or DCA of reducing agent and hindering the further growth having luminescent properties of quantum clusters with size of 10-22 nm and shows UV emission at 300-335 nm, no plasma resonance and 2nd order visible emission at 590-650 nm resemble to the nanoparticles or super critical nucleus are mentioned.
However recently, numerous studies have shown that size and capping agent of nanoparticles play an important role in cellular uptake and cytotoxicity; (B. Devika Chithrani and W. C. W. Chan; ACS Nanolett. 2007, 7(6), 1542-1550; Yu Pan; Willi Jahnen Dechent; Small, 2007, 3(11), 1941-1949 and Catherine J. Murphy; J Nanopart Res; 2010, 12, 2313-2333); X. D. Zhang et al; biomaterials; 2012, 33, 6408-6419) they have also shown that 10-27 nm size range of AuNP are most biocompatible and having negligible toxicity. Another problem with the small gold nano crystals is toxicity and difficulty in handling the very small size nano clusters. It was reported that 1.4 nm diameter particles were toxic, whereas 15 nm diameter particles were nontoxic, even at up to 100-fold higher concentrations. Particles above 5 nm are non fluorescent. Nanorod's are having the SPR and also the luminescence near IR and potential candidates for photothermal therapy against cancer but shows the toxicity by formation of reactive oxygen species (Nicole M. Schaeubin et al ACS, langmuire 2012, 28, 3248-3258). QD's are excellent probe due to distinctive quantum confinement effect and tunable and high photo luminescence however its toxic effects are of serious concern especially for bioapplications. (Francoise M. Winnik et al; ACS accounts of chemical research; 2013, 46 (3), 672-680).
Walter H. Chang et al; J. Medcal and biological Engg.; 2009, 29(6), 276-283 shows the blue, green and red emission by using the various capping agent and etching the NP's and final size of these nanocluster lies in the range of 1-3.5 nm however being very small they may lend the toxic effect.
Huan-Tsung Chang et al; Anal Chem.; 2008, 80, 1497-1504 synthesized 2.9 nm brightly luminescent MUDA capped Au nanodots which are stable for three months when stored in dark and at 4° C. Review by Didier Astruc et al; Chemi rev.; 2004, 104; 293-346 showed the aggregation of MUDA capped AuNP in acidic condition. C. A. J. Lin et al ESBME-Peter; AuNP capped with MUDA using tetraborate buffer, 9.2 pH. AuNP's 3.1 nm capped with MUDA shows the agglomeration when synthesized at room temperature as compared the cold synthesis (Mark T. Swihart, Colloids and Surfaces A: Physicochem Eng. Aspects 2004. 246; 109-113).
Alexander Gaiduk et al; publication no. WO 2012028936 A1 and application number: PCT/IB2011/002003 showed the green and red emission by using the organic solvent and photo thermal microscopy showed the enhancement in fluorescence in solid state. They coated the glass surface with glycerol. Fluorescence was not observed when water was used. However we do not have any such kind of restrictions, instrumentation and no photo-thermal therapy was needed. The particles are fluorescent in both solid and liquid state.
These problems are of a great challenge if the AuNP's are to be used in biological systems especially in vivo applications. Hence, there have been many attempts to tailor make the process.
Aniruddha S. Deshpande et al; Nanoscale res. lett.; 2008, 3, 221-229; synthesized Sulphur nanoparticles with an average size of 10 nm using the oil phase and 15 (vol. %) aqueous phase contains iron chelate and the H2S gas was used as a precursor.
Jun Lin et al; materials letters; 2001, 49, 282-286; synthesized AuNP using 0.056 M HAuCl4 and 0.32 M NaBH4 using CTAB/octane reverse micelle and stabilized with dodecanethiol resulted in the formation of 1D, 2D and 3D superstructures.
Even after continuous research there are long standing problems limiting the full utilization of AuNC's like uniform size clusters, their thermo dynamical stability as clusters are having the tendency to grow further. Moreover, once the clusters reach to critical size they formed supercritical nucleus and grow further until the growth is arrested by capping agent. Once they form the supercritical nucleus they loose the property of luminescence and surface plasmon is dominated. Another difficulty is water soluble and isolable nanocrystals/nanoparticles (Kyosti Kontturi et al; ChemPhysChem; 2006, 7, 2143-2149). We have overcome both the hurdles; the 10-22 nm globular aggregates stabilized with amine group of hydrazine hydrate or dicarboxyacetone of citric acid or capped with mercaptopropionic acid or MUDA can be easily extracted by centrifugation and re-dispersed in water. Stability at low pH is desirable for drug delivery especially AuNP's capped with MUDA, mercaptoundeconic acid which in general are stable only in highly basic pH 9-13. Huan-Tsung Chang (Anl. Chem. 2008, 80, 1497-1504) showed stabilization at low pH by reducing tempt. to 4° and storing in dark; is again a limitation. The stability at low pH 3.5 MUDA capped 10-22 nm globular nano aggregates has the potential to replace the toxic PEG/bifunctional PEG (high cost) which at presently used for the drug delivery and many other bio-applications, also shows some form of toxicity. (Xiao-Dong Zhang et al). The 10-22 nm globular aggregates can be used dynamically in various bio applications including drug delivery.
These problems make the use of gold nanocrystals/nano dots restrictive in biological systems, especially for in vivo applications. Further, according to some studies Ramsay D L, Arch Dermatol. 1992 July; 128(7):931-3 possible protective role for UV-β therapy has been suggested in case of cutaneous T-cell lymphoma and immuno-regulatory. UV-β irradiating in geriatric patients increase the level of 25-hydroxyvitamin D levels (B. L. Diffy, Phys. Med. Biol.; 1980, V. 25 (3), 405-426). UV-β therapy was also used for skin disorders like lupus and sepsis; against infection of antibiotics resistance strains etc (Dr. Jonathan V. Wright: Harnessing the healing power of light Part 1). In such cases also role of desirable size of AuNP's/AuNC's or AgNP/AgNC's with selective UV-β luminescence is contemplated wherein it may be applied locally on the affected area.
Hence, there is a long standing need of prior art of AuNC's of controlled aggregate of 10 nm to 22 nm size which are biocompatible, non-toxic, capable of UV-β and visible emissions which may be used dynamically for various biomedical applications like UV and, phototherapy, drug delivery, biosensing, bio-labelling/bioimaging applications simultaneously. Also, the fluorescence remains unchanged in solid state for both as synthesized amine/DCA capped as well as after capping with MUDA/MPA.
The emission energy decreases with increasing number of atoms. The inventor has attempted to overcome the limitations of prior art and disclosed narrow size distributed, photo stable, multicolour fluorescence and capped globular nano aggregates (of size 10-22 nm) made up of 2-6 atoms ultra small clusters (encapsulated in a matrix of amine/dicarboxyacetone) with the functional group amine or carboxyl extractable and dispersed in water; capable of UV emission at 300-335 nm+/−5 nm; visible emissions: green emission when blue/green filter was used and 590-650 nm 2nd order red emission; no plasmon resonance at 500-550 nm. These 10-22 nm size aggregates of atomic quantum ultra small nanoclusters which by virtue of the larger aggregate size (10-22) are safer than individual atomic nanoclusters/nanoparticles of 1-5 nm (Yu Pan, Willi Jahnen Dechent; Small, 2007, 3(11), 1941-1949), while at the same time retaining the optical properties of fluorescence which is the characteristics of atomic quantum 1-5 nm nanoclusters/nanoparticles (Beatriz Santiago Gonzalze et al: ACS Nanolett. 2010, 10, 4217-4221). Such aggregates can be the excellent fluorescent probe including for cell imaging both in vivo and in vitro, sensing and therapy. By virtue of the intense fluorescence which can replace the toxic semiconductor quantum dots etc.